INTRODUCTION:

Understanding the structure and function of the skin, also known as the cutaneous membrane, is important for healthcare professionals. In adults, skin covers 1.2 to 2.2 m² and includes hair-bearing skin on most of the body and hairless skin on the palms and soles. The skin is the most exposed body part, facing sunlight, environmental pollution, and providing some protection against pathogens^1,2,3.

Over 80% of the global population still relies heavily on traditional medicines for treating various skin diseases⁴. Recently, there has been a revival of interest in medicinal plants in developing countries due to their safety and relatively fewer side effects compared to synthetic drugs^5,6. Topical herbal treatments have gained significant attention because of their widespread use and favourable benefit/risk ratio⁷. According to Fortune Business Insights, the global herbal medicine market is projected to grow from $165.66 billion in 2017 to $347.50 billion by 2029, at a CAGR of 11.16%. India has 45,000 plant species, with 15,000-20,000 used medicinally. The domestic medicinal plant market was Rs. 4.2 billion (US$ 56.6 million) in 2019 and is expected to reach Rs. 14 billion (US$ 188.6 million) by 2026⁸.

Body wash is used for cleansing and maintaining body hygiene. It helps prevent the growth of bacteria, acne, and cleanse impurities like oil, dirt, makeup, and sweat. Liquid body wash is more convenient, affordable, hygienic, and easier to transport and store than solid soap, making it popular with the general population⁹. Natural bath soaps are rare, with synthetic ingredients like triclosan, common in antibacterial soaps, potentially irritating skin and causing thyroid issues. Many manufacturers are now using natural ingredients for safer, non-irritating soaps¹⁰.

Acne vulgaris, affecting 95% of boys and 83% of girls, is a chronic inflammatory disorder impacting areas with large oil glands like the face, back, and trunk. It features seborrhea, comedones, inflammatory lesions, and bacteria such as Propionibacterium acnes and Staphylococcus aureus. Increased sebum production leads to acne, with P. acnes damaging skin cells and promoting S. aureus growth. Overuse of antibiotics has caused bacterial resistance, prompting exploration of medicinal plants as alternative treatments^11,12.

Recent studies highlight the potential of Ashwagandha (Withania somnifera) and Senna alata in dermatology, particularly for topical applications targeting antibacterial, anti-inflammatory, and anti-acne benefits. Ashwagandha, known for its withanolides, demonstrates considerable antimicrobial and anti-inflammatory effects, potentially reducing acne severity and supporting skin elasticity. Clinical trials on formulations containing Ashwagandha extract have shown improvement in parameters like hydration, elasticity, and a reduction in transepidermal water loss, making it beneficial for maintaining skin barrier integrity and countering skin aging¹³.

Senna alata, rich in anthraquinones like chrysophanol and rhein, has shown promise for its antimicrobial and antifungal properties, proving effective against pathogens like Staphylococcus aureus, commonly involved in skin infections. Studies of Senna alata extract in topical formulations reveal potential in reducing skin infections, suggesting it could play a preventive role against acne-related bacterial growth¹⁴.

Both herbs show promise when incorporated into body washes, offering a synergistic effect that could enhance skin health by balancing sebum production, supporting skin barrier function, and providing an anti-inflammatory action. Combining these extracts in formulations like liquid body washes can thus leverage their antimicrobial and skin-soothing properties, filling a need for herbal-based topical solutions in skincare.

This study examines two medicinal plants traditionally used for their antimicrobial activity against microorganisms frequently involved in acne inflammation, such as Propionibacterium acnes and Staphylococcus aureus. Senna alata and Ashwagandha extracts, rich in secondary metabolites, have been used to treat skin diseases. While many studies have focused on their pharmacological properties, fewer have explored their use in pharmaceutical and cosmetic formulations, such as creams, oils, and soaps. Currently, no combination of Ashwagandha and Senna alata exists in a liquid body wash on the market. This study aims to develop a polyherbal liquid body wash with anti-acne and antibacterial properties using these extracts, addressing a gap in research and product availability.

MATERIALS AND METHODS:

Chemicals:

HEC was purchased from Sd fine chem limited Mumbai, HPMC, Muller hinton agar/broth, DMSO was purchased Hi media Mumbai, Glycerin, PVPK-30 was purchased from Moly-chem Mumbai, Coco glucoside and Decyl glucoside, Fragrance oil was purchased from purenso Indore, HPLC grade methanol, Methanol, ethanol was purchased HPLC grade water from Merck Germany.

Plant collection and authentication:

Ashwagandha roots were purchased from Hebsur Store in Hubballi, and Senna alata leaves were gifted by Bio Crops, Visakhapatnam, the plants were authenticated and certified by Prof. Dr Subhas N. Emmi from H.S. Kothambari Science Institute, Hubballi.

Extraction of Ashwagandha root:

Dried Ashwagandha roots were powdered after drying. 10g of this powder was mixed with 100ml of methanol and macerated for 24hours with continuous agitation to obtain the extract. The resulting methanolic extract was then evaporated to dryness in a water bath, yielding a semi-solid residue stored in a desiccator for further use¹⁵.

Extraction of Senna Alata leaf:

Dried Senna alata leaves were powdered using mortar and pestle, sieved to uniform size, and loaded into the Soxhlet extraction apparatus. Soxhlet extraction was performed in 3 batches using 25g of Senna alata powder and 750ml of 80% ethanol per batch. Extraction continued for 10 hours at 50°C until the color of the leaves turned green to brown. The resulting extract underwent simple distillation to separate the crude extract from the ethanolic solvent. The crude extract with traces of water was evaporated in a china dish to obtain a thick, semi-solid extract, which was then weighed to calculate the percentage yield¹⁶.

In-vitro Anti-microbial activity for Senna Alata leaf and Ashwagandha root extract.

Antimicrobial activity was tested in three batches using the well diffusion technique. Staphylococcus aureus was streaked on Muller Hinton agar plates and Propionium Bacterium on Thioglycolate agar plates. Wells were created using a 6mm sterile borer. Senna alata leaf extract (10-50mg/ml) and methanolic Ashwagandha root extract (100-500 mg/ml) were dissolved in DMSO. An accurately measured 100µl each were added to the wells. Ciprofloxacin was the positive control, and DMSO was the negative control. Plates were left for 1 hour at room temperature for diffusion, and then incubated at 37°C for 24-48 hours. Clear zones of inhibition indicating antibacterial activities were measured and averaged^17,18,19.

Qualitative analysis of Ashwagandha and Senna alata extract by TLC:

TLC analysis utilized Silica Gel 60 F254 plates 3.5×1.5 cm for Ashwagandha and 4.3×5cm plates for Senna Alata. For Ashwagandha, 1µL of standard sample was applied from 100 mg substance dissolved in 10mL methanol. The mobile phase consisted of chloroform and methanol in a 9:1 ratio, and 5% methanolic sulphuric acid was sprayed as a detector, with spot detection at 254nm. For Senna alata, 100mg of the sample was dissolved in 10 mL DMSO and a standard markers concentration of 1mg/mL were used. The mobile phase was a mixture of ethyl acetate: methanol: water in a 4:4:3 ratio, and 10% methanolic KOH was sprayed for detection at 254nm^16,20.

Extract-excipient compatibility by FTIR analysis:

Extract-excipient compatibility was assessed using Shimadzu FTIR Affinity 1S spectroscopy in a topical polyherbal liquid body wash formulation. FTIR spectra of the individual extract and the formulation were compared in the range of 4000-300 cm⁻¹ to ensure safety and efficacy.

Preparation of polyherbal liquid body wash:

The polyherbal liquid body wash was formulated using the contents as shown in table 1. Firstly, the gelling agent HEC and HPMC were sonicated in water for 30 minutes, and PVP K-30 was added. In another beaker, coco glucoside, decyl glucoside, glycerin, fragrance oil and remaining water were mixed thoroughly and added to the prepared gelling agents. Senna alata leaf extract and Ashwagandha root extract at 1% and 2% concentrations were incorporated into the placebo liquid body wash formulation using a mortar and pestle to obtain uniform polyherbal liquid body wash.

Table no 1: Formulation table of Polyherbal liquid body wash

Ingredients	HEC F1	HEC F2	HPMC F3	HPMC F4
Hydroxy ethyl Cellulose	3.5g	3.5g	--	--
Hydroxy propyl methyl cellulose	-	-	3.0g	3.0g
PVP k-30	1.2g	1.2g	1.2g	1.2g
Coco glucoside	5ml	5ml	5ml	5ml
Decyl glucoside	5ml	5ml	5ml	5ml
Glycerine	3ml	3ml	3ml	3ml
Fragrance oil	0.25ml	0.25ml	0.25ml	0.25ml
Water	32ml	32ml	32ml	32ml
Ashwagandha extract	1%	2%	1%	2%
Senna alata extract	1%	2%	1%	2%

Antimicrobial sensitivity test for formulations of Senna alata and Ashwagandha polyherbal liquid body wash by well diffusion technique:

The well diffusion technique was used to test microbial sensitivity of the polyherbal liquid body wash formulations. A 6mm sterile borer made wells in each petri dish. Three batches of formulations (1% and 2% each of Senna alata and Ashwagandha extracts) were tested. 100µl of each formulation was added to Mueller Hinton agar plates for Staphylococcus aureus and Thioglycollate agar plates for Propionium bacterium. Plates were left for 1 hour at room temperature for diffusion, then incubated at 37°C for 24 - 48hours. Staphylococcus aureus was incubated aerobically and Propionium bacteria anaerobically. Zones of inhibition indicating antibacterial activities were measured and averaged^17,18,19.

Evaluation parameters of Polyherbal liquid body wash:

Determination of Physicochemical parameters:

The physical appearance, homogeneity, and washability of the formulations were evaluated. The organoleptic test observed the color, odor, and texture. Homogeneity was checked by applying the preparation on glass or transparent materials to detect any particle specks, with none indicating a homogeneous mixture. Washability was assessed by applying a small amount of the liquid body wash to the hand and rinsing it with tap water to ensure easy washability^21,22,23,24.

Determination of PH:

The pH of the placebo and polyherbal liquid body wash formulations was measured using a HANNA digital pH meter. After calibrating the pH meter with buffer solutions of pH 7 and pH 10, one gram of each sample was diluted in 100ml of distilled water and measured at room temperature^25,26.

Foam stability test:

The foam stability test for the polyherbal liquid body wash and placebo were performed by diluting 1g of the sample in 10ml of distilled water in a test tube. The solution was shaken by turning the test tube back and forth, and the initial foam level was recorded. The tube was then left undisturbed for 5 minutes to measure the retained foam height^27,28

Final Foam Height

Foam stability = --------------------------- X 100

Initial Foam Height

Determination of foamability:

To determine foamability, 1g of the polyherbal liquid body wash or placebo was dissolved in 50ml of water in a beaker, shaken vigorously 10 times, and transferred to a 100ml measuring cylinder. The foam volume was then read directly from the calibrated scale²⁹.

Viscosity:

Rheological studies of the polyherbal liquid body wash and placebos were conducted for 3 batches using a Brookfield viscometer. After allowing 30g of the liquid body wash to settle in a beaker for 5 minutes, spindle TL7 was attached and dipped into the sample. The viscometer motor was turned on, and readings were recorded at 0.6, 1.0, 1.5, and 2.0 rpm^30,31.

Stability studies:

Stability studies for the optimized formulations of the polyherbal liquid body wash were conducted according to ICH guidelines at 30°C±2°C and 65%±5% RH over one month. The formulations were stored in glass bottles and evaluated on the 1st, 7th, 15th, and 30th days. Parameters like physical appearance, pH, foam stability, foamability, viscosity, and antimicrobial activity were examined in triplicate³².

RESULTS AND DISCUSSIONS:

Extraction of Ashwagandha root and Senna alata leaf:

Extraction was performed in three batches, each with 10g of crude Ashwagandha root powder in 100ml of methanol for 24hours, yielding approximately 90ml of crude extract. This extract was heated at 80°C to remove methanol, resulting in a thick semi-solid extract with a final yield of 0.9g and an average percentage yield of 9.4%. Similarly, extraction containing 25g of crude Senna alata leaf powder in 750ml of ethanol was carried out for 10hours, producing approximately 700ml of extract. This was distilled to obtain 150ml of crude extract by removing ethanol. The crude extract was then heated at 80°C to remove water, yielding a thick semi-solid extract with a final yield of 4.6g and an average percentage yield of 18.4%. The yields were tabulated in Table 2.

Table no: 2 Ashwagandha root and Senna alata leaf extraction process

Sample	Average percentage yield (%)
Methanolic Ashwagandha root extract	9.4%
80% ethanolic Senna alata leaf extract	18.4%

Antimicrobial sensitivity test for 80% ethanolic Senna alata leaf extract and methanolic root extract of Ashwagandha using bacterial strain Staphalococcus aureus and Propionium bacterium:

The antibacterial activity of 80% ethanolic Senna alata leaf extract and methanolic Ashwagandha root extract against Staphylococcus aureus and Propionibacterium acnes was investigated. As shown in Fig 1(A). the Senna alata leaf extract showed inhibition zones ranging from 13.6mm to 20.0mm against Staphylococcus aureus, with the highest inhibition at 50mg/ml (20.0mm) and the lowest at 10mg/ml (13.6mm) and Fig 1(B) the Ashwagandha root extract displayed inhibition zones from 17.6mm to 25.3mm, with the highest at 500mg/ml (25.3mm) and the lowest at 100mg/ml (17.6mm). Ciprofloxacin, the positive control, exhibited the highest inhibition at 37mm (0.2mg/ml), while DMSO, the negative control, showed no activity. Against Propionibacterium acnes, as shown in Fig 2(A) Senna alata extract exhibited inhibition zones from 9.3mm to 17.6 mm, with the highest at 4mg/ml (17.6mm) and no inhibition at 1mg/ml. Whereas, the Ashwagandha extract showed zones from 5.6mm to 18.6mm, with the highest at 4mg/ml (18.6mm) and the lowest at 1mg/ml (5.6mm) shown in Fig 2(B). Ciprofloxacin again showed the highest inhibition (37mm at 0.2mg/ml), while DMSO showed no activity. The results were tabulated in Table 3. Therefore, Ashwagandha demonstrates greater overall potency than Senna alata, especially against Staphylococcus aureus, where its inhibition levels are notably higher. The results indicate that Ashwagandha’s active components, such as withanolides, may provide stronger antibacterial effects compared to the anthraquinones in Senna alata. However, Senna alata’s effectiveness, particularly at its higher concentrations, still supports its use as a complementary antimicrobial, potentially enhancing Ashwagandha’s effects when combined. This analysis underscores the potential synergistic role these two extracts could play in formulations targeting acne-related and general skin infections.

Figure no 1: Antimicrobial sensitivity testing for bacterial strain Staphylococcus aureus (A) Senna alata leaf extract, (B) Ashwagandha root extract

Figure no 2: Antimicrobial sensitivity testing for bacterial strain Propionium bacteria (A) Senna alata leaf extract (B) Ashwagandha root extract

Qualitative Analysis of TLC for Senna alata leaf and Ashwagandha root extract:

The ethanolic Senna alata leaf extract found out that Rhein, emodin and aloe-emodin were the chemical constituents that showed antimicrobial effects and constituent withaferin A in methanolic Ashwagandha root extracts. Ashwagandha root (L) were analyzed using mobile phase chloroform: methanol (9:1) and found to be the best in giving clear and well resolved spots for methanolic root extract of Ashwagandha. The results showed 0.31 Rf value for the sample and 0.32 for standard (with reference to article), both the values are nearer to each other, it indicates the presence of Withaferin A in the tested sample as shown in table no 4 and Fig 3(B). Many runs were performed to qualitatively analyse the separation of 80% Senna alata leaf extracts using thin layer chromatography (TLC). Mobile phase n-propanol: ethyl acetate: water (40:40:30 v/v/v) was used for separation and observed the presence of few compounds as shown in Figure no 3(A). The Rf values for the sample and standards were calculated and tabulated in Table no 4. The compounds were found to be major constituents with Rf values 0.5 for rhein and 0.8 for aloe-emodin. The results can be seen visually and using, UV spectrophotometers with wavelengths of 254 nm.

Table no 4: Qualitative analysis of Withania Somnifera root extract and Senna alata leaf extract by TLC

SI. no	Sample	UV Detector	Sample RF Value	Standard RF value
1.	Withania Somnifera	254nm	0.31	0.32
2.	Senna Alata	254nm	0.5, 0.86	0.5 Rhein, 0.8 Aloe-emodin, 0.9 Emodin

A B

Figure no 3: TLC of A) Senna alata leaf at UV at 254nm B) Ashwagandha root Extracts at UV at 254nm

Extract-excipient compatibility test by FTIR analysis:

The FTIR analysis confirmed the presence of various functional groups such as hydroxyl, alkyl, carbonyl, alcohol, ether, alkane, aryl ether, methoxy, and amines. As shown in Fig 4 It showed good compatibility between the excipients and extracts used in the preparation of the liquid body wash.

Fig no 4: FTIR Spectrum of polyherbal extracts and its formulations

Preparation of topical liquid body wash containing Senna alata leaf and Ashwagandha root extract:

Polyherbal formulations of liquid body wash were developed following pre-formulation studies. Wherein, FTIR spectroscopy was performed to choose the excipients based on their compatibility with the extract. This comprehensive approach ensured the stability, safety, and efficacy of the resulting polyherbal liquid body wash formulation. The excipients used in the formulation were tabulated in table 1.

In vitro Antimicrobial sensitivity test for polyherbal formulation of extracts for bacterial strain Staphylococcus aurues and Propionium bacteria:

The antimicrobial sensitivity test was performed using the well diffusion technique with Staphylococcus aureus and Propionium bacterium acne for the prepared polyherbal formulations, as shown in Figures 5(A), 5(B), 5(C) and 5(D). For Staphalococcus aureus Figure 5(A) showed the average zone of inhibition 23mm to 21mm for polyherbal formulation and no activity for placebo containing HPMC. Whereas, Figure 5(B) depicts the average zone of inhibition 24mm to 22mm for polyherbal formulation and 10mm inhibition was observed for placebo containing HEC. For Propionium bacteria Fig 5(C) showed the average zone of inhibition 13.6mm to 11.6mm for polyherbal formulation and 6.3mm activity for placebo containing HPMC. Whereas, Figure 5(D) showed the average zone of inhibition 13.66mm to 10.3mm for polyherbal formulation and 5.6mm inhibition for placebo containing HEC. The polyherbal formulation with 2% Senna alata leaf extract and Ashwagandha root extract showed maximum antibacterial activity for Staphalococcus aureus and Propionium bacterium. The zone of inhibition was increased as there was increase in concentrations. And formulations of polyherbal liquid body wash showed better inhibition compared to leaf extract and less inhibition was observed for the placebo formulation compared to polyherbal formulation. The results were tabulated in table 5

Table no 5: Antimicrobial activity of liquid body wash on Staphylococcus aureus and Propionium bacteria

*Staphalococcus aureus*	Formulation using HPMC		Formulation using HEC
	1%	21mm	1%	22mm
	2%	23mm	2%	24mm
	-ve	00	-ve	10mm
*Propionium* bacteria	1%	11.66mm	1%	10.33mm
	2%	13.66	2%	13.66mm
	-ve	6.33	-ve	5.66

Fig no 5: Antimicrobial activity of polyherbal formulation containing Senna alata leaf extract and Ashwagandha root extract of liquid body wash against bacterial strain Staphylococcus aureus A) HPMC, B) HEC and Propionium bacteria C) HPMC D) HEC

Evaluation parameters of Polyherbal liquid body wash:

Determination of physicochemical parameters:

The physical characteristics of the polyherbal liquid body wash were visually examined, including color, odor, and texture, with placebo formulations showing an amber-yellow color and pleasant odor, while polyherbal formulations were darkish brown with a pleasant fragrance. All formulations were semisolid, smooth, and homogeneous, with no phase separation observed. Washability was excellent, leaving no traces after washing off with tap water.

Determination of pH:

As tabulated in table 6, the pH of both placebo and polyherbal formulations met the range of 5.5 to 7.5. The average pH was 7.2 for placebo formulation and the average pH for the formulation of polyherbal liquid body wash was in the range of 7 to 7.4.

Foam Stability studies:

Foam stability, indicating the resistance of soap bubbles to maintain size, was measured after 5 minutes and recorded in Table 6. Placebo formulations showed foam stability of 76.5% for HEC and 77.8% for HPMC, while Polyherbal formulations ranged from 60% to 80%, all within the acceptable range.

Foamability Studies:

The volume of foam produced, was in accordance with the standard acceptance range of >15cm (Table 6). The placebo formulations had foamability of 15.3 cm (HEC) and 14cm (HPMC), while polyherbal formulations ranged from 15 to 18 cm.

Viscosity Studies:

Viscosity was measured using a Brookfield viscometer; it adhered to the standard specification of 400-4000 cps. Table 6 lists viscosities: placebo HEC at 4217.1 cps (0.6 rpm) and 1478.8 cps (2.0rpm); placebo HPMC at 2723.06 cps (0.6rpm) and 537.76 cps (2.0 rpm); polyherbal HEC F1 at 5999.3 cps (0.6rpm) and 2806.7 cps (2.0rpm); HEC F2 at 4005.1 cps (0.6 rpm) and 1623.1 cps (2.0rpm); HPMC F3 at 6290.3 cps (0.6 rpm) and 3501.1 cps (2.0rpm); and HPMC F4 at 3823.1 cps (0.6rpm) and 1000.7 cps (2.0rpm).

Stability studies:

Optimized polyherbal liquid body wash formulations stored at 30°C±2°C and 65%±5% RH for 3 months. The physical appearance, colour, odour, PH, foamability, foam stability, viscosity, antimicrobial test was in the acceptable limit and both the formulations F2 and F4 was stable. This indicates that the formulation polyherbal liquid body wash was stable at stability studies and confirms that the formulations had no interaction based on climatic conditions like temperature, humidity, and light.

Table no 6: Evaluation of parameters of Polyherbal formulations of liquid body wash:

Evaluation parameters	Placebo formulation HPMC	Placebo formulation HEC	Polyherbal Formulations
Evaluation parameters	Placebo formulation HPMC	Placebo formulation HEC	F1	F2	F3	F4
Colour	Amber yellow	Amber yellow	Brown	Brown	Brown	Brown
Texture	Smooth	Smooth	Smooth	Smooth	Smooth	Smooth
Odour	Pleasant	Pleasant	Pleasant	Pleasant	Pleasant	Pleasant
Phase separation	No phase separation	No phase separation	No phase separation	No phase separation	No phase separation	No phase separation
Homogeneity	Homogeneous	Homogeneous	Homogeneous	Homogeneous	Homogeneous	Homogeneous
Washability	Good	Good	Good	Good	Good	Good
pH	7.2	7.26	7.32	7.36	7.41	7.45
Foam Stability	76.5	77.8	62.2	72.4	63.9	76.1
Foamability	15.3	14.2	15.3	16.6	14.8	18.2
Viscosity	4217.1-1478.8	2723.06 - 537.76	5999.3 - 2806.7	4005.1 - 1623.1	6290.03 - 3501.1	3823.1 - 1000.7

CONCLUSION:

The study focused on developing a topical herbal remedy for acne and bacterial infections using Senna alata and Ashwagandha extracts. Following standardization, antimicrobial activity was tested through zone of inhibition assays, revealing that increased concentrations enhanced inhibition against Staphylococcus aureus and Propionibacterium acnes. TLC confirmed active compounds like aloe-emodin, rhein, and Withaferin A in the extracts. Formulation optimization and stability studies confirmed the effectiveness of a polyherbal liquid body wash, with a brown color, slight basic pH, and favorable foam stability, foamability, and viscosity. Compatibility testing and FTIR spectroscopy supported the formulation's stability and suitability. Stability testing over three months at 30°C and 65% RH showed no degradation, validating the body wash’s resilience. In conclusion, this polyherbal formulation demonstrates strong potential for consumer use as a natural acne treatment. Key findings underscore its effectiveness, stability, and safe properties. Future research, including long-term stability and skin irritation tests, could further establish the product's efficacy and widen its practical applications.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank KLE Academy of Higher Education and Research, Belagavi for their financial support during the lab studies.

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Received on 21.06.2024 Revised on 26.10.2024

Accepted on 30.01.2025 Published on 12.06.2025

Available online from June 14, 2025

Research J. Pharmacy and Technology. 2025;18(6):2708-2715.

DOI: 10.52711/0974-360X.2025.00389

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

*Staphalococcus aureus*	*Senna alata leaf Extract*		*Ashwagandha* root extract
	Concentration (mg/ml)	Inhibition (in mm)	Concentration (mg/ml)	Inhibition (in mm)
	10	13.6	100	17.66
	20	16.0	200	23
	30	17.3	300	24
	40	18.6	400	24.6
	50	20.0	500	25.3
	Positive control (Ciprofloxacin 0.2mg /ml)	37	Positive control (Ciprofloxacin 0.2mg /ml)	37
	-ve Control (DMSO)	00	-ve Control (DMSO	00
*Propionium bacteria*	1	00	1	5.66
	2	9.33	2	11.33
	3	15.33	3	16.66
	4	17.66	4	18.66
	-ve control	00	-ve control	00